Effects of ketone body 3-hydroxybutyrate on cardiac and mitochondrial function during donation after circulatory death heart transplantation

Normothermic regional perfusion (NRP) allows assessment of therapeutic interventions prior to donation after circulatory death transplantation. Sodium-3-hydroxybutyrate (3-OHB) increases cardiac output in heart failure patients and diminishes ischemia–reperfusion injury, presumably by improving mitochondrial metabolism. We investigated effects of 3-OHB on cardiac and mitochondrial function in transplanted hearts and in cardiac organoids. Donor pigs (n = 14) underwent circulatory death followed by NRP. Following static cold storage, hearts were transplanted into recipient pigs. 3-OHB or Ringer’s acetate infusions were initiated during NRP and after transplantation. We evaluated hemodynamics and mitochondrial function. 3-OHB mediated effects on contractility, relaxation, calcium, and conduction were tested in cardiac organoids from human pluripotent stem cells. Following NRP, 3-OHB increased cardiac output (P < 0.0001) by increasing stroke volume (P = 0.006), dP/dt (P = 0.02) and reducing arterial elastance (P = 0.02). Following transplantation, infusion of 3-OHB maintained mitochondrial respiration (P = 0.009) but caused inotropy-resistant vasoplegia that prevented weaning. In cardiac organoids, 3-OHB increased contraction amplitude (P = 0.002) and shortened contraction duration (P = 0.013) without affecting calcium handling or conduction velocity. 3-OHB had beneficial cardiac effects and may have a potential to secure cardiac function during heart transplantation. Further studies are needed to optimize administration practice in donors and recipients and to validate the effect on mitochondrial function.

Infusion of amiodarone (10 μg/kg/min) and a bolus of 100 mg lidocaine were administered to stabilize heart rhythm during the experiment.Both donor and recipient received 1.5 g Cefuroxime and 1000 mg Solu-Medrol at the start of the procedure.Plasma concentrations of 3-OHB were measured using a point of care device (Freestyle Precision Neo, Abbott, USA).

Donor procedure
Premedication was administered at the housing facility before transport to the surgical facilities.
Via the right femoral artery, an aortic occlusion balloon was advanced to the abdominal aorta above the iliac bifurcation.After sternotomy, umbilical tapes were placed around the aortic arch vessels and inferior vena cava.A Pressure-volume catheter was inserted in the left ventricle via the left carotid artery.The standard roller pump CPB (cardiopulmonary bypass) circuit was primed with 1000 mL Ringers lactate, 200 mL mannitol and 100 mL 8.4% sodium bicarbonate.

DCD, NRP and Intervention
Functional warm ischemic time (fWIT) was defined as the time from systolic blood pressure <50 mmHg to the onset of NRP.Circulatory arrest was defined as central venous pressure (CVP) = mean arterial pressure (MAP).Three minutes before NRP, the abdominal aortic balloon was inflated and pulled distally to the iliac bifurcation and aortic arch vessels ligated.NRP was commenced for 60 min, and the ventilation was restarted with a FiO2 60% and titrated to PaO2 levels between 12-16 kPa.Norepinephrine was used to maintain MAP >60 mmHg during NRP.At 5, 15 and 55 min after onset of NRP, arterial and mixed venous blood samples were collected.Biochemical parameters including arterial pH, K + , and glucose were corrected during NRP.Fifteen min before weaning from NRP, infusion of dobutamine (2.5 μkg/kg/min) was started and the heart gradually loaded and weaned from NRP.

Recipient procedure
Anesthesia for the recipient animals were maintained with propofol (3.5 mg/kg/h) and fentanyl (15 μg/kg/h).Baseline hemodynamic measurements were performed before start of infusion of 3-OHB or placebo for the duration of the experiment.Systemic anticoagulation with 40.000IU heparin and ascending aortic and bi-caval cannulation were used to institute CPB.Flow rates were adjusted to MAP > 60 mmHg and normothermia was maintained throughout the recipient procedure.
The order of anastomosis was left atrium, inferior vena cava, superior vena cava followed by 250 mL antegrade HTK cardioplegia before pulmonary artery and finally aorta were anastomosed.A vent was placed in the apex of the LV and de-airing maneuvers were performed before removal of the aortic cross-clamp and reperfusion of the donor heart.Internal defibrillation (30 J) was used to treat ventricular arrhythmias.Dobutamine (1.5 -2.5 μg/kg/min) and norepinephrine (0.05 -1.0 μg/kg/min), were used for inotropic and hemodynamic support, respectively.The hearts were gradually volume-loaded and CPB flow decreased; weaning was considered successful if the animals maintained a MAP above 50 mmHg for 30 min after discontinuation of CPB.
The permeabilized fiber bundles were added to the chambers in the Oxygraph-2k, filled with 2mL MiR05 at 37°C.
To evaluate physiological mitochondrial respiration, the following substrateuncoupler-inhibitor-titration (SUIT) protocol was used: Glutamate (10 mmol/L) and Malate (2 mmol/L) without presence of ADP stimulate basal complex I respiration.Subsequent addition of ADP (5 mmol/L) allows complex I mediated respiration with electron flow through the mitochondrial ATPase (GM3).Succinate (10 mmol/L) was added to stimulate maximal respiration with electron flow through complex I+II (GMS3).Oligomycin (complex V inhibitor) (2 µg/mL) was added to evaluate state 4o leak respiration.Final addition of rotenone (complex I inhibitor) (0.5 µmol/L) and antimycin A (complex III inhibitor) (2.5 mmol/l) allows measurement of residual oxygen consumption.
To evaluate respiration driven by oxidative phosphorylation of ß-hydroxybutyric acid, the following SUIT protocol was used: Glutamate (10 mmol/L) and Sodium-3-OHB (in titration steps to reach maximal respiration) stimulates basal mitochondrial respiration without ADP.Subsequent addition of ADP (5 mmol/L) allows evaluation of respiration through complex I. Final addition of succinate (10 mmol/L) stimulates maximally coupled respiration with electron flow through complex I+II.
To avoid any O2 limitations to respiration the chambers were hyperoxygenated and all measurements were carried out in duplicate.The integrity of the outer mitochondrial membrane was tested by the addition of cytochrome c (10 µmol/L).An increase of >10% compared with complex I mediated coupled respiration (GM3) in the oxygen consumption rate led to exclusion.
The data were corrected for residual oxygen consumption and the OXPHOS capacity was calculated as respiration for complex I+II subtracted with leak state respiration 1 .We calculated the respiratory control ratio (RCR) as maximal respiration (GMS3 for glucose linked substrates or GBS3 for 3-OHB substrates) divided by leak state (GM or GOHB).

In vitro hiPSC study
Human fibroblasts were reprogrammed by retroviral delivery of three reprogramming factors to obtain the patient-specific human induced pluripotent stem cells (hiPSCs) as previously described (SOX2, KLF4 and OCT4) 2,3 .Undifferentiated hiPSC were grown in mTeSR Plus (Stemcell technologies, Vancouver, Canada) on Matrigel coated plates and passaged every 4 days at 1:10 ratio using 5mM EDTA solution (Life Technologies, California, USA).

Micro-Organoid Tissue engineering
Human iPSCs derived cardiomyocytes grown as monolayers were detached using TrypLE express X1 for 5 minutes at 37C⁰.The detached cardiomyocytes were seeded on a V-bottom 96 well plate (Thermo Fisher, Massachusetts, USA) in a concentration of 10 4 cells/50µL for each well.
After 7 days, video recordings were carried out using a robotic inverted microscope (Olympus IX83, Olympus, USA).The organoids were video recorded for 8 seconds (for each well) in a Okolab Incubator (Italy) at 37C⁰ and in the presence of 5% CO2.

Micro-Organoid Tissue Analysis
All signals shorter than 200ms and longer than 600ms were excluded.Outliers were calculated using a Python script that calculates the quantiles, IQR, and the upper and lower limitation.The outlier signals were excluded from the calculation of the average of the micro-organoid signal.
Bazett's formula was used for calculating the contraction duration and relaxation time (calibrating for the variability in the beating rate).The total number of wells that were included in the analysis are 24 wells of 3-OHB and 21 wells of NaCl.
After 7-10 days, the cells were loaded with the calcium indicator Fluo-4 AM (Thermo Fisher, Massachusetts, USA).Cells were incubated with 1µL Fluo-4/1mL of RPMI-B27 for 30 minutes and were measured in Tyrode's solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 10 mM glucose; pH 7.4).Fluorescent signal changes were acquired in a line-scan mode for optical-signal analysis for depicting changes in cell-fluorescence.Four wells of each group (control and 3-OHB) were included in the analysis.